Description:
Initiation of DNA replication is highly regulated so that it occurs no more than once per cell cycle. DNA amplification overrides these cellular controls, and extra copies of DNA are produced at specific loci. DNA amplification is a hallmark of cancer, but the mechanism underlying the initiation of DNA amplification is poorly understood. To elucidate how DNA amplification starts, we are studying developmentally-regulated, locus-specific DNA amplification in the late larval salivary gland giant polytene chromosomes of the fungus fly Sciara coprophila. Polytene chromosomes result from the entire genome undergoing multiple rounds of endoduplication without intervening mitosis. Locus-specific DNA amplification is superimposed on the last endoduplication cycle to create DNA puffs visible under a light microscope. Transcription follows amplification; however, at one well-characterized puff site (DNA puff II/9A), RNA polymerase II (Pol II) is present at the onset of amplification, which is 48 hours before active transcription. It is also known that the binding of the ecdysone receptor (a steroid hormone receptor that acts as a transcription factor) to this locus induces amplification, suggesting a link between the promotion of transcription and of amplification. This raises the question of the purpose that Pol II serves at the amplification stage and how it is prevented from transcribing until amplification is finished. NELF (Negative Elongation Factor) is a protein known in Drosophila to stall RNA polymerase and prevent transcription elongation. My study aims to elucidate the genome-wide extent of the early presence of Pol II and asks whether Pol II might be held back from transcribing by NELF while DNA amplification takes place. I have approached this question by using immunofluorescence with an antibody against NELF-B and correlation with previous immunofluorescence results showing the genome-wide spatio-temporal localization of the ecdysone receptor and active transcription on the polytene chromosomes. Pol II, where present, may open up the chromatin structure to allow access for the machinery for DNA replication/amplification as well as the machinery for transcription. This would provide an explanation of the numerous origins of DNA replication that map to the promoters of genes, as well as a link between DNA replication and transcription.